It is generally recognized that to be effective as antibacterial agents the plasma level of aminoglycosides must be maintained at above about 2 .mu.g./ml. At concentration greater than about 10 or 12 .mu.g./ml. these antibiotics exhibit a number of serious toxic side effects in that at such levels they can be ototoxic (auditory impairment) or nephrotoxic (renal function impairment), particularly with neonates and those exhibiting renal problems prior to treatment (see Smith et al, New England Journal of Medicine, Vol. 302, pp. 1106-1109 (1980)). In come rare cases these antibiotics have even been implicated in neural blockades. As a consequence, close monitoring of the serum aminoglycoside concentration is desirable so that the concentration can be generally kept within the 2-12 .mu.g./ml. therapeutic range to allow for the effective treatment of bacterial infection while minimizing the possible toxic side effects.
Various assays have been developed and adopted to protect patients and to ensure effective antibacterial doses. Up to the present invention, however, such assays have been very expensive, inaccurate, laborious, require expensive equipment or require long assay times. In addition, such methods have tended to be specific to a particular aminoglycoside, have required the handling of radioisotopes or have been subject to interference by other compounds in the serum. Examples of such methods are: the bioassay, wherein the serum sample is tested microbiologically against sensitive bacterial strains through observation of zones of inhibition against bacterial growth; radioimmunoassay and radioenzymatic assays, which require specialized equipment and the handling of radioisotopes; latex agglutination, which is expensive and technically laborious; Emit method which requires purchase of an expensive kit and software package.
In addition to the above described assay methods a potential colorimetric method has also been known. This method finds a theoretical basis in the following equations: ##EQU1##
TNB.sup..sym. is an intensely yellow colored compound which can be readily measured spectrophotometrically at 412 nm.
Although offering attractive possibilities, the method represented by the above schematic has not been applied to biological fluid samples, and particularly not serum (blood) samples, since DTNB will react with any sulfhydryl groups (proteins) represented by X--SH in the sample and since no enzyme was available which was characterized by a high specific activity against aminoglycosides and which would bring about reaction A rapidly.